About 40% of the women diagnosed with breast cancer will progress to metastatic disease. However, metastases cannot be removed by surgery or radiation, and most metastases are chemoresistant. Therefore, therapies that specifically prevent or eliminate metastases offer great promise in the outcome. Recent studies indicate that enhancement of specific helper or cytotoxic T lymphocyte (CTL) responses to breast tumors through vaccination could potentially lead to the specific elimination of micro-metastases. However, poor vaccine-induced immune and clinical responses underscore the need for improved vaccine strategies. This research proposal is focused on the improvement of vaccine therapy against metastatic breast cancer by combining vaccination with the IL-6 inhibitor curcumin, using the preclinical metastatic mouse breast tumor model 4T1. We previously demonstrated that Mage-b DNA vaccination provides significant protection against breast cancer metastases in this model, albeit not completely. Interleukin (IL)-6 may contribute to decreased vaccine efficacy. It is highly up regulated in the 4T1 primary tumors and metastases, probably via the Nuclear Factor Kappa B (NFkB), and is a potent regulator of dendritic cells (DC) differentiation in vivo. IL-6 activates the expression of transducer and activator of transcription (STAT)3 in immature DC, preventing the DC from maturation and subsequent presentation of antigens to T cells. This may lead to T cell unresponsiveness. Agents that are able to inhibit IL-6 may lead to enhanced vaccine efficacy. Curcumin could be such an agent, since it inhibits IL-6 production. Both Mage-b and IL-6 are frequently detected in human breast cancer, which makes the 4T1 model clinically relevant. The long-term objective of this proposal is to develop a novel non- toxic combination therapy of Mage-b vaccination and curcumin in the 4T1 model, with a focus on its translation into human clinical trials, if successful. The hypothesis is that curcumin improves T cell activation and subsequent immune responses upon Mage-b DNA vaccination, resulting in further reduction of the frequency of breast cancer metastases. The specific aims are as follows:(1) Testing the effect of Mage-b vaccination with and without curcumin on tumors and metastases. For this purpose, mice will be immunized preventively or therapeutically with Mage-b DNA vaccine and challenged with 4T1 tumor cells. As soon as the primary tumor can be felt curcumin will be administered daily. Frequency of metastases, tumor size, and survival times will be determined;(2) Testing the direct effect(s) of curcumin on vaccine- and tumor-induced immune responses, and on proliferation and apoptosis of tumor cells. For this purpose, spleen, lymph nodes, tumors and metastases of treated and control mice will be analyzed for the presence of activated CD4 and CD8 T cells, regulatory T cells and mature DC. In addition, tumors and metastases of treated and control mice will also be analyzed for the expression of NFkB and NFkB-regulated genes that are directly involved in immune responses such as IL-6, TNF1, and STAT3, and for proliferation and apoptosis. PUBLIC HEALTH RELEVANCE: Current treatment of cancer is ineffective against metastases. In this research proposal a novel non-toxic combination therapy of Mage-b vaccination and curcumin will be developed that is effective against breast cancer metastases, using a preclinical mouse model that reflects metastatic breast cancer in humans.